Studies were initiated to investigate the genetic stability of the genes of the cold-adapted (ca) influenza A/Ann Arbor/60 donor virus that confer attenuation on reassortant viruses. Initially, a single gene reassortant was prepared that contained the PB2 gene of the ca donor virus in a background of 7 other genes derived from a human wild type influenza A virus. The PB2 protein is a constituent of the viral polymerase complex that also includes the PB1 and PA proteins. The PB2 gene from tile ca donor virus specifies a temperature sensitivity (ts) phenotype. ts+ revertants were selected and analyzed genetically. Four independent ts+ revertants developed a suppressor ("second site") mutation in the PA gene. In contrast, sequence analysis indicated that the sequence of the mutant PB2 gene was unaltered. These observations suggest that a suppressor mutation developed in another protein of the polymerase complex (i.e., PA) of the 4 revertants and this mutation corrected the defect in polymerase activity specified by the mutant PB2 gene. Coding or non-coding mutations in attenuating genes occur infrequently during the process of producing live attenuated influenza A virus vaccine by gene transfer from an attenuated donor virus to a reassortant virus bearing the surface glycoproteins of a wild type human influenza A virus. The NP and M genes, which are the attenuating genes of the A/Mallard/78 avian influenza A donor virus, are genetically stable after prolonged replication of influenza A virus reassortants in primates or humans. A single gene reassortant containing the PB2 gene of the attenuated avian influenza A/Mallard/78 donor virus and the other 7 RNA segments derived from the virulent human A/LA/87 (H3N2) virus was shown to be markedly restricted in replication in mammalian cells, but this reassortant replicated efficiently in avian cells. Studies are in progress to determine if this gene constellation will consistently attenuate wild type human influenza A viruses for monkeys and humans.